Project Summary T lymphocyte dysfunction underlies autoimmunity, chronic infection, and cancer. Breakthrough therapies that reprogram defective T cells represent one of the most promising strategies for overcoming these monumental health challenges. This has primarily been accomplished by modulating signal transduction in T cells through the use of antibodies and recombinant cytokines that promote specific T cell functional programs. While there has been major progress in understanding the signaling pathways and the epigenetic mechanisms that govern T cell reprogramming, the biochemical architecture that supports these processes has largely been overlooked. Advances in the field of cancer biology have revealed cell metabolism plays an active regulatory role in cell signaling and epigenetics, but it is unclear how stereotyped or varied this process is in a diverse and dynamic system like the immune system. I have generated preliminary data showing that mitochondrial metabolism regulates epigenetic remodeling during helper T (Th) cell differentiation in a Th cell program specific manner, such that individual metabolic processes influence specific histone lysine modifications depending on differentiating conditions. As outlined in this proposal, I will set out to systematically delineate the molecular mechanisms that directly explain this metabolic-epigenetic axis during T cell reprogramming. I will employ a Cas9 screening system I have developed and used with great success in deconvoluting how metabolic networks support primary immune cell activation and function. By targeting individual metabolic enzymes with CRISPR, we will seek to elucidate how mitochondrial metabolism and Warburg metabolism influence histone methylation and acetylation. We will then identify the downstream epigenetic modifying enzymes that are responsible for the observed phenotype by performing epistasis/rescue studies whereby we target both the metabolic enzyme and the candidate epigenetic modifier with tandem sgRNA expression vectors. These in vitro findings will then be validated using in vivo transfer models with CRISPR targeted T cells. To expand on the findings from my postdoctoral work, I will also take a second parallel approach, where I will aim to discover novel metabolic pathways that regulate epigenetic remodeling in differentiating T cells. To accomplish this, I will take advantage of a sgRNA library I created and already successfully used to study how metabolism controls T cell activation, in which every gene for a metabolic enzyme or transporter is targeted by CRISPR. I will use with this metabolome sgRNA library to identify metabolic pathways necessary for H3K9 and H3K27 acetylation and methylation, unique to Th17 or Treg conditions. These studies will not only elucidate how metabolism controls cell transcriptional programming by setting the biochemical environment in which epigenetic remodeling occurs, but will also inform the design of new, low cost metabolism-based therapies for immune cell reprogramming.